The present invention relates to novel forms of vascular endothelial growth factor genes and the novel proteins encoded by these genes. More particularly, the invention relates to novel forms of human VEGF-A. These novel forms of VEGF-A include VEGF-A138, VEGF-A162, and VEGF-A182. Such novel VEGF proteins may be used in the treatment of the cardiovascular system and its diseases through effects on anatomy, conduit function, and permeability, and more particularly in the treatment of cardiovascular disease by stimulating vascular cell proliferation using a growth factor, thereby stimulating endothelial cell growth and vascular permeability.
The invention also relates to nucleic acids encoding such novel VEGF proteins, cells, tissues and animals containing such nucleic acids; methods of treatment using such nucleic acids; and methods relating to all of the foregoing.
Cardiovascular diseases are generally characterized by an impaired supply of blood to the heart or other target organs. Myocardial infarction (MI), commonly referred to as heart attacks, is a leading cause of mortality as 30% are fatal in the first months following the heart attack. Heart attacks result from narrowed or blocked coronary arteries in the heart which starves the heart of needed nutrients and oxygen. When the supply of blood to the heart is compromised, cells respond by generating compounds that induce the growth of new blood vessels so as to increase the supply of blood to the heart. These new blood vessels are called collateral blood vessels. The process by which new blood vessels are induced to grow out of the existing vasculature is termed angiogenesis, and the substances that are produced by cells to induce angiogenesis are the angiogenic factors.
Unfortunately, the body""s natural angiogenic response is limited and often inadequate. For this reason, the discovery of angiogenic growth factors has lead to the emergence of an alternative therapeutic strategy which seeks to supplement the natural angiogenic response by supplying exogenous angiogenic substances.
Attempts have been made to stimulate angiogenesis by administering various growth factors. U.S. Pat. No. 5,318,957 to Cid et al. discloses a method of stimulating angiogenesis by administering haptoglobins (glyco-protein with two polypeptide chains linked by disulfide bonds). Intracoronary injection of a recombinant vector expressing human fibroblast growth factor-5 (FGF-5) (i.e., in vivo gene transfer) in an animal model resulted in successful amelioration of abnormalities in myocardial blood flow and function. (Giordano, F. J., et. al. Nature Med 2, 534-539, 1996). Recombinant adenoviruses have also been used to express angiogenic growth factors in-vivo. These included acidic fibroblast growth factor (Muhlhauser, J., et. al. Hum. Gene Ther. 6:1457-1465, 1995), and one of the VEGF forms, VEGF-A165 (Muhlhauser, J., et. al. Circ. Res. 77:1077-1086, 1995).
One of the responses of heart muscle cells to impaired blood supply involves activation of the gene encoding Vascular Endothelial Growth Factor (xe2x80x9cVEGFxe2x80x9d), also known as VEGF-A, (Banai, S., et. al. Cardiovasc. Res. 28:1176-1179, 1994). VEGF-A is actually a family of angiogenic factors that induce the growth of new collateral blood vessels. These growth factors are specific angiogenic growth factors that have vaso-permeability activity and target endothelial (blood vessel-lining) cells almost exclusively. (Reviewed in Ferrara et al., Endocr. Rev. 13:18-32 (1992); Dvorak et al., Am. J. Pathol. 146:1029-39 (1995); Thomas, J. Biol. Chem. 271:603-06 (1996)). Expression of the VEGF-A gene is linked in space and time to events of physiological angiogenesis, and deletion of the VEGF-A gene by way of targeted gene disruption in mice leads to embryonic death because the blood vessels do not develop. It is therefore the only known angiogenic growth factor that appears to function as a specific physiological regulator of angiogenesis.
When tested in cell culture, VEGF-A, and, because of its structure, likely VEGF-B, (VEGF""s) are potently mitogenic (Gospodarowicz et al., Proc. Natl. Acad. Sci. USA 86:7311-15, 1989) and chemotactic (Favard et al., Biol. Cell 73:1-6, 1991). Additionally, VEGFs induce plasminogen activator, plasminogen activator inhibitor, and plasminogen activator receptor (Mandriota et al., J. Biol. Chem. 270:9709-16, 1995; Pepper et al., 181: 902-06, 1991), as well as collagenases (Unemori et al., J. Cell. Physiol. 153:557-62, 1992), enzyme systems that regulate invasion of growing capillaries into tissues. VEGFs also stimulate the formation of tube-like structures by endothelial cells, an in vitro example of angiogenesis (Nicosia et al., Am. J. Pathol., 145:1023-29, 1994).
In vivo, VEGFs induce angiogenesis (Leung et al., Science 246:1306-09, 1989) and increase vascular permeability (Senger et al., Science 219:983-85, 1983). VEGFs are now known as important physiological regulators of capillary blood vessel formation. They are involved in the normal formation of new capillaries during organ growth, including fetal growth (Peters et al., Proc. Natl. Acad. Sci. USA 90:8915-19, 1991), tissue repair (Brown et al., J. Exp. Med. 176:1375-79, 1992), the menstrual cycle, and pregnancy (Jackson et al., Placenta 15:341-53, 1994; Cullinan and Koos, Endocrinology 133:829-37, 1993; Kamat et al., Am. J. Pathol. 146:157-65, 1995). During fetal development, VEGFs appear to play an essential role in the de novo formation of blood vessels from blood islands (Risau and Flamme, Ann. Rev. Cell. Dev. Biol. 11:73-92, 1995), as evidenced by abnormal blood vessel development and lethality in embryos lacking a single VEGF allele (Carmeliet et al., Nature 380:435-38, 1996). Moreover, VEGFs are implicated in the pathological blood vessel growth characteristic of many diseases, including solid tumors (Potgens et al., Biol. Chem. Hoppe-Seyler 376:57-70, 1995), retinopathies (Miller et al., Am. J. Pathol. 145:574-84, 1994; Aiello et al., N. Engl. J. Med. 331:1480-87, 1994; Adamis et al., Am. J. Ophthalmol. 118:445-50, 1994), psoriasis (Detmar et al., J. Exp. Med. 180:1141-46, 1994), and rheumatoid arthritis (Fava et al., J. Exp. Med. 180:141-46, 1994).
VEGF expression is regulated by hormones (Schweiki et al., J. Clin. Invest. 91:2235-43, 1993) growth factors (Thomas, J. Biol. Chem. 271:603-06, 1996), and by hypoxia (Schweiki et al., Nature 359:843-45, 1992, Levy et al., J. Biol. Chem. 271:2746-53, 1996). Upregulation of VEGFs by hypoxic conditions is of particular importance as a compensatory mechanism by which tissues increase oxygenation through induction of additional capillary vessel formation and resulting increased blood flow. This mechanism is thought to contribute to pathological angiogenesis in tumors and in retinopathies. However, upregulation of VEGF expression after hypoxia is also essential in tissue repair, e.g., in dermal wound healing (Frank et al., J. Biol. Chem. 270:12607-613, 1995), and in coronary ischemia (Banai et al., Cardiovasc. Res. 28:1176-79, 1994; Hashimoto et al., Am. J. Physiol. 267:H1948-H1954, 1994).
Using the rabbit chronic limb ischemia model, it has been shown that repeated intramuscular injection or a single intra-arterial bolus of VEGF-A can augment collateral blood vessel formation as evidenced by blood flow measurement in the ischemic hindlimb (Pu, et al., Circulation 88:208-15, 1993; Bauters et al., Am. J. Physiol. 267:HI263-71, 1994; Takeshita et al., Circulation 90 [part 2], II-228-34, 1994; Bauters et al., J. Vasc. Surg. 21:314-25, 1995; Bauters et al., Circulation 91:2802-09, 1995; Takeshita et al., J. Clin. Invest. 93:662-70, 1994). In this model, VEGF has also been shown to act synergistically with basic FGF to ameliorate ischemia (Asahara et al., Circulation 92:[suppl 2], II-365-71, 1995). VEGF was also reported to accelerate the repair of balloon-injured rat carotid artery endothelium while at the same time inhibiting pathological thickening of the underlying smooth muscle layers, thereby maintaining lumen diameter and blood flow (Asahara et al., Circulation 91:2793-2801, 1995). VEGF has also been shown to induce EDRF (Endothelin-Derived Relaxin Factor (nitric oxide))-dependent relaxation in canine coronary arteries, thus potentially contributing to increased blood flow to ischemic areas via a secondary mechanism not related to angiogenesis (Ku et al., Am. J. Physiol 265:H586-H592, 1993).
Activation of the gene encoding VEGF-A results in the production of several different VEGF-A variants, or isoforms, produced by alternative splicing wherein the same chromosomal DNA yields different mRNA transcripts containing different exons thereby producing different proteins. Such variants have been disclosed, for example, in U.S. Pat. No. 5,194,596 to Tischer et al. which identifies human vascular endothelial cell growth factors having peptide sequence lengths of 121, and 165 amino acids (i.e., VEGF-A121 and VEGF-A165). Additionally, VEGF-A189 and VEGF-A206 have also been characterized and reported (Neufeld, G., et. al. Cancer Metastasis Rev. 15:153-158, 1996).
The mitogenic activity of the various VEGF-A isoforms varies depending on each isoform. For example, VEGF-A121 and VEGF-A165 have very similar mitogenic activity for endothelial cells. However, VEGF-A189 and VEGF-A206 are only weakly mitogenic (Ferrara et al., Endocr. Rev. 13:18-32, 1992). The reduced activity of these isoforms is attributed to their strong association with cells and matrix, as evidenced by the normal mitogenic activity of a mutant of VEGF-A206 which lacks the 24-residue xe2x80x9cmatrix targetingxe2x80x9d sequence common to VEGF-A189 and VEGF-A206 (residues 115-139 in FIG. 2) (Ferrara et al., Endocr. Rev. 13:18-32, 1992).
Four known forms of VEGF-A arise from alternative splicing of up to eight exons of the VEGF-A gene (VEGF-A121, exons 1-5, 8; VEGF-A165, exons 1-5,7,8; VEGF-A189, exons 1-5, 6a, 7, 8; VEGF-A206, exons 1-5, 6a, 6b, 7, 8 (exon 6a and 6b refer to 2 alternatively spliced forms of the same exon)) (Houck et al., Mol. Endocr., 5:1806-14 (1991)). All VEGF-A genes encode signal peptides that direct the protein into the secretary pathway. For example, VEGF-A165 cDNA encodes a 191-residue amino acid sequence consisting, of a 26-residue secretary signal peptide sequence, which is cleaved upon secretion of the protein from cells, and the 165-residue mature protein subunit. However, only VEGF-A121 and VEGF-A165 are found to be readily secreted by cultured cells whereas VEGF-A189 and VEGF-A206 remain associated with the producing cells. These VEGF-A forms possess an additional highly basic sequence encoded by exon 6 corresponding to residues 115-139 in VEGF-A189 and residues 115-156 in VEGF-A206. These additions confer a high affinity to heparin and an ability to associate with the extracellular matrix (matrix-targeting sequence) (Houck, K. A. et al., J. Biol. Chem. 267:26031-37 (1992) and Thomas, J. Biol. Chem, 271:603-06 (1996)). The mitogenic activities of VEGF-A121 and VEGF-A165 are similar according to the results of several groups (Neufeld, G., et al., Cancer Metastasis Rev. 15:153-158 (1996) although one research group has shown evidence indicating that VEGF-A121 is significantly less active (Keyt, B. A., et al. J. Biol. Chem. 271:7798-7795 (1996). It is unclear whether the two longer VEGF-A forms, VEGF-A189 and VEGF-A206, are as active or less active than the two shorter forms since it has not been possible to obtain them in pure form suitable for quantitative measurements. This failure is due in part to their strong association with producing cells and extracellular matrices which is impaired by the presence of exon-6 derived sequences apparently acting in synergism with exon-7 derived sequences groups (Neufeld, G., et al., Cancer Metastasis Rev. 15:153-158 (1996).
As depicted in FIG. 1, the domain encoded by exons 1-5 contains information required for the recognition of the VEGF receptors flt-1 (R1 and R2) and KDR/flk-1 (Keyt, B. A., et. al. J. Biol Chem 271:5638-5646, 1996), and is present in all known VEGF isoforms. The amino-acids encoded by exon 8 are also present in all known isoforms. The isoforms may be distinguished however by the presence or absence of the peptides encoded by exons 6 and 7 of the VEGF-A gene, and the presence or absence of the peptides encoded by these exons results in structural differences which are translated into functional differences between the VEGF-A forms (reviewed in: Neufeld, G., et. al. Cancer Metastasis Rev. 15, 153-158. 1996).
Exon 6 can terminate after 72 bp at a donor splice site wherein it contributes 24 amino acids to VEGF forms that contain it such as VEGF-A189. This exon 6 form is referred to herein as exon 6a. However, the VEGF-A RNA can be spliced at the 3xe2x80x2 end of exon 6 using an alternative splice site located 51 bp downstream to the first resulting in a larger exon 6 product containing 41 amino-acids. The additional 17 amino-acids added to the exon 6 product as a result of this alternative splicing are referred to herein as exon 6b. VEGF-A206 contains the elongated exon 6 composed of 6a and 6b, but this VEGF form is much rarer than VEGF-A189. (Tischer, E., et al., J. Biol. Chem. 266, 11947-11954; Houck, K. A., et al., Mol. Endocrinol., 1806-1814, 1991).
A putative fifth form of VEGF-A, VEGF-A145, has been noted in the human endometrium, using PCR. The authors state that the sequence of the cDNA of the VEGF-A145 splice variant indicated that it contained exons 1-5, 6 and 8. However, it is uncertain whether the authors found that the splice variant contained exons 6a and 6b as in VEGF-A206, exon 6a as in VEGF-A189, or exon 6b. The authors state that since the splice variant retains exon 6 it is probable that it will be retained by the cell as are the other members of the family that contain this exon. (Charnock-Jones et al., Biology of Reproduction 48, 1120-1128 (1993); see also, Bacic M., et al. Growth Factors 12, 11-15, 1995). The biologic activity of this form was not established in that report (Cheung, C. Y., et al., Am. J. Obstet. Gynecol., 173, 751-759, 1995); Anthony, F. W. et al., Placenta, 15, 557-561, 1994). The various isoforms, and the exons that encode the isoforms, are depicted in FIG. 1.
More recently, a VEGF-A protein of 145 amino acids, and nucleic acid encoding this protein, have been identified, and its use for treating the cardiovascular system and its diseases has been identified (U.S. Ser. No. 08/784,551 now U.S. Pat. No. 6,013,780, filed Jan. 21, 1997, published as WO 98/10071 on Mar. 12, 1998).
VEGF-A is known to bind to two of three different endothelial cell receptors, each of which is a single transmembrane protein with a large extracellular portion comprised of 7 immunoglobulin-type domains and a cytoplasmic portion that functions as a tyrosine kinase. These receptors are R1 (flt-1) (De Vries et al., Science 255:989-91, 1992), R2 (KDR/flk-1) (Terman et al., Biochem. Biophys. Res. Commun. 187:1579-86, 1992), and R3 (flk-4) (Pajusola et al., Cancer Res. 52:5738-43, 1992). There are distinct selectivities between these receptors and the various VEGF-A and VEGF-related protein ligands that have not been completely elucidated as yet. However, it is known that VEGF-A binds to R1 and R2 (Terman et al., Growth Factors 11:187-95, 1994) but not R3 (Joukov et al., EMBO J. 15:290-98, 1996). R2 is thought to be primarily responsible for the angiogenic response of endothelial cells to VEGF-like growth factors (Gitay-Goren et al., J. Biol. Chem. 271:5519-23 (1996)).
Accordingly, there is a need for new forms of VEGF-A proteins that have modified affinities for matrix and low affinity receptor. This modified affinity will alter its bioavailability when administered.
The present invention is directed to novel forms of VEGF proteins, preferably human VEGF-A proteins. The preferred use of the VEGF proteins and nucleic acid molecule compositions of the invention is to use such compositions to treat the cardiovascular system and its diseases through effects on anatomy, conduit function, and permeability. Such proteins and compositions may be used to aid in the treatment of patients with heart disease, wounds, or other ischemic conditions by stimulating angiogenesis in such patients. These novel forms of VEGF proteins will have a modified affinity for matrix and low affinity receptors. This modification alters the bioavailability of the proteins when administered directly to cells or when cells are transduced with DNA encoding these proteins.
These novel VEGF proteins will possess a unique combination of biological properties that will distinguish them from other VEGF forms. The unique combination of properties of these VEGF proteins will render them preferred therapeutic agents in certain circumstances for the treatment of the cardiovascular system and its diseases as well as other diseases characterized by vascular cell proliferation. In particular, the cDNA coding for these VEGF proteins may be employed in gene therapy for treating the cardiovascular system and its diseases.
The novel VEGF-A proteins comprise amino acid sequences coded for, for example, by VEGF-A exons 1-5, 6b, and 8, or 1-5, 6b, 7, and 8 or a derivative thereof. These proteins preferably do not comprise the full amino acid sequence of exon 6a and, preferably, do not have the same properties, activity, and function of the corresponding VEGF-A proteins that comprise the full amino acid sequence of exon 6a. Other novel VEGF-A proteins comprise amino acid sequences coded for, for example, by VEGF-A exons 1-5, 6a, 6b, and 8. These proteins preferably do not have the same properties, activity, and function of the corresponding VEGF-A proteins that do not comprise exon 6b.
Thus, in preferred embodiments of the invention, a purified polypeptide is provided, comprising an amino acid sequence coded for by VEGF-A exons 1-5, 6b, and 8, or a derivative thereof. In other preferred embodiments of the invention, a purified polypeptide is provided comprising an amino acid sequence coded for by VEGF-A exons 1-5, 6b, 7, and 8, or a derivative thereof.
In other preferred embodiments of the invention, a purified polypeptide is provided comprising an amino acid sequence coded for by VEGF-A exons 1-5, 6a, 6b, and 8, or a derivative thereof. Preferably, the VEGF-A polypeptide of the invention is human VEGF-A. In preferred aspects, the purified polypeptide comprises the amino acid sequence of FIG. 3, FIG. 4, or of FIG. 5.
Also provided in the invention are purified and isolated nucleic acid molecules coding for the purified polypeptides of the invention. Preferably, the nucleic acid molecules have the nucleotide sequence of FIG. 3 or FIG. 4, or of FIG. 5.
Also provided in the present invention are purified and isolated nucleic acid molecules coding for a biologically active fragment of a 6b-modified VEGF protein, preferably VEGF-A138, VEGF-A162, or VEGF-A182, or a derivative thereof. Such fragments maintain most preferably up to 100% of the activity of the full length 6b-modified and have at least 10%, more preferably at least 40%, more preferably at least 80% of the activity of the full length VEGF-A138, VEGF-A162, or VEGF-A182 polypeptides.
Also provided are expression vectors comprising the nucleic acid molecules of the invention. Preferably, these vectors comprise adenovirus sequences; preferably the expression vector is an adenovirus vector. More preferably, the nucleic acid is operably linked to a promoter sequence that is active in vascular endothelial cells. The expression vector preferably further comprises a partial adenoviral sequence from which the E1A/E1B genes have been deleted.
In another embodiment of the invention, a kit is provided for intracoronary injection of a recombinant vector expressing a 6b-modified protein, preferably VEGF-A138, VEGF-A162, or VEGF-A182, comprising: a nucleic acid molecule encoding a 6b-modified protein, preferably VEGF-A138, VEGF-A162, or VEGF-A182, cloned into a vector suitable for expression of said polynucleotide in vivo, a suitable container for said vector, and instructions for injecting said vector into a patient. Preferably, in the kit, the polynucleotide is cloned into an adenovirus expression vector.
In yet another aspect of the invention, a method is provided for treating vascular disease in a mammal comprising the step of administering to said mammal a 6b-modified VEGF-A protein, preferably, VEGF-A138, VEGF-A162, or VEGF-A182, or other 6b-modified VEGF-A protein in a therapeutically effective amount to stimulate vascular cell proliferation.
In another aspect of the invention, a method is provided for enhancing endothelialization of diseased vessels comprising the step of administering to a mammal a therapeutically effective amount of a 6b-modified protein, preferably VEGF-A138, VEGF-A162, or VEGF-A182. Preferably, the endothelialization is reendothelialization after angioplasty. More preferably, the reendothelialization reduces or prevents restenosis.
In further aspects of these methods of the invention, the patient is treated with or without a stent. Preferably, the mammals used in the methods of the invention are human, however, it is contemplated that all mammals would be candidates for these methods. In the methods of the invention, the administration may comprise gene therapy. In preferred methods, the gene for gene therapy is administered using an inflatable balloon catheter coated with a polynucleotide encoding 6b-modified VEGF protein, preferably VEGF-A138, VEGF-A162, or VEGF-A182.
In yet other preferred embodiments of the present invention, the methods, compositions, and vectors of the invention may be used to enhance drug permeation by tumors comprising administering to a patient a 6 b-modified VEGF-A protein or a nucleic acid molecule coding for a 6b-modified VEGF-A protein, preferably VEGF-A138, VEGF-A162, or VEGF-A182. The 6b-modified VEGF-A protein may be delivered directly to a tumor cell, or it may be delivered into the vascular system, preferably at a site located close to the site of the tumor. Thus, delivery of the 6b-modified VEGF-A protein in conjunction with chemotherapy to remove or reduce the size of a tumor, will help to enhance the effectiveness of the chemotherapy by increasing drug uptake by the tumor. The 6b-modified VEGF-A protein delivered in this method may either be through direct delivery of the polypeptide or protein, or through gene therapy.
Thus, provided in the present invention is a method of enhancing drug permeation by tumors comprising administering to a patient a nucleic acid molecule coding for a 6b-modified VEGF-A protein, preferably VEGF-A138, VEGF-A162, or VEGF182. Preferably, the 6b-modified VEGF-A protein is delivered directly into a tumor cell.
In another aspect of the invention, a therapeutic composition is provided comprising a pharmaceutically acceptable carrier and a 6b-modified VEGF protein, preferably VEGF-A138, VEGF-A162, or VEGF182, in a therapeutically effective amount to stimulate vascular cell proliferation. Also provided as an aspect of the invention is a filtered injectable adenovirus vector preparation, comprising: a recombinant adenoviral vector, said vector containing no wild-type virus and comprising: a partial adenoviral sequence from which the E1A/E1B genes have been deleted, and a transgene coding for a 6b-modified VEGF protein, preferably VEGF-A138, VEGF-A162, or VEGF-A182, driven by a promoter flanked by the partial adenoviral sequence; and a pharmaceutically acceptable carrier.
In further embodiments of the invention are provided methods for treating cardiovascular disease in a mammal comprising the step of transfecting cells of said mammal with a nucleic acid molecule which encodes an amino acid sequence coded for by VEGF-A exons 1-5, 6b and 8. Also provided are methods for treating cardiovascular disease in a mammal comprising the step of transfecting cells of said mammal with a nucleic acid molecule which encodes an amino acid sequence coded for by VEGF-A exons 1-5, 6b, 7, and 8. Also provided are methods for treating cardiovascular disease in a mammal comprising the step of transfecting cells of said mammal with a nucleic acid molecule which encodes an amino acid sequence coded for by VEGF-A exons 1-5, 6a, 6b, and 8.
Preferably, the nucleic acid molecule employed in the methods of the invention codes for VEGF-A138, VEGF-A162, or VEGF-A182. In preferred embodiments, the nucleic acid molecule is cloned into a vector. Preferably, the vector comprises adenovirus particles. Preferably, the adenovirus vector particles are delivered to said mammal by injection. Preferably, the number of said adenovirus particles is between about 108 to about 1014, more preferably between about 1010 to about 1014.
In further preferred aspects, the polynucleotide which encodes a 6b-modified VEGF protein, preferably VEGF-A138, VEGF-A162, or VEGF-A182, is delivered to the heart of a mammal. Intracoronary delivery of the polynucleotide is preferred, preferably according to the methods set forth in PCT/US96/02631, published Sep. 6, 1996 as WO96/26742, hereby incorporated by reference herein. Preferably, an intracoronary injection is conducted about 1 cm into the lumens of the left and right coronary arteries.
Thus, in preferred embodiments, the transfected cells are muscle cells, including but not limited to myoblasts, myocytes, cardiomyocytes, cardioblasts, and smooth muscle cells. Preferably, the cells are cardiomyocytes. More preferably, the transfected cells are coronary artery cells and said injection is intracoronary injection. More preferably, the adenovirus particles are injected at about 1 cm into the lumens of the left and right coronary arteries.
Preferably, these methods further comprise administering a potentiating agent that potentiates the angiogenic effect of the 6b-modified VEGF-A polypeptide. Preferably, the potentiating agent is an angiogenic FGF. More preferably, the potentiating agent is selected from the group consisting of FGF-1, FGF-2, FGF-4, FGF-5, and FGF-6.
In one preferred aspect of the methods of the invention, the cells are transfected in vivo. In other preferred aspects, the said cells are transfected ex vivo.
Endothelial cell proliferation, such as that which occurs in angiogenesis, is also useful in preventing restenosis following balloon angioplasty. The balloon angioplasty procedure often injuries the endothelial cells lining the inner walls of blood vessels. Smooth muscle cells often infiltrate into the opened blood vessels causing a secondary obstruction in a process known as restenosis. The proliferation of the endothelial cells located at the periphery of the balloon-induced damaged area in order to cover the luminal surface of the vessel with a new monolayer of endothelial cells would potentially restore the original structure of the blood vessel.
Thus, the present invention provides a method of treating cardiovascular disease in a mammal comprising the step of transfecting cells of said mammal with a polynucleotide which encodes a 6b-modified VEGF-A protein, preferably VEGF138, VEGF-A162, or VEGF-A182. In preferred aspects, the polynucleotide is cloned into a vector. In further preferred aspects, the vector is an adenovirus vector. The adenovirus vector is preferably delivered to the mammal by injection; preferably, about 108 to about 1014 adenovirus vector particles are delivered in the injection. More preferably, about 1011 to about 1013 adenovirus vector particles are delivered in the injection. Most preferably, about 1012 adenovirus vector particles are delivered in the injection.
Thus in further preferred embodiments, the nucleic acid molecule is introduced into the coronary artery by a catheter inserted into said artery. Preferably, the catheter comprises an inflatable balloon having an outer surface adapted to engage the inner wall of said artery, and wherein said nucleic acid molecule is disposed upon said balloon outer surface.
In preferred aspects of the methods of the invention, the nucleic acid molecule comprises the nucleotide sequence of FIG. 3, FIG. 4, or FIG. 5.
In other aspects of the invention, transformed or transfected host cells are provided which comprise the expression vectors of the invention. Such host cells may be used in, for example, methods of producing a VEGF-A polypeptide comprising growing, under suitable conditions, a host cell transformed or transfected with the recombinant DNA expression vector of the invention in a manner allowing expression of said polypeptide, and isolating said polypeptide from the host cell.
Also provided in the present invention is a method of treating a patient suffering from an ischemic condition comprising administering a therapeutic amount of a pharmaceutical composition comprising a 6b-modified VEGF protein, preferably VEGF-A138, VEGF-A162, or VEGF-A182, in a suitable carrier. Preferably, this method further comprises administering an agent that potentiates the therapeutic effect of said 6b-modified VEGF-A polypeptide. Preferably, the potentiating agent is selected from the group consisting of FGF-1, FGF-2, FGF-4, FGF-5, and FGF-6. Further, said ischemic condition is preferably selected from the group consisting of: cardiac infarction, chronic coronary ischemia, chronic lower limb ischemia, stroke, and peripheral vascular disease.
For the treatment of peripheral conditions, such as peripheral vascular disease, administration of the pharmaceutical compositions of the present invention is preferably by delivery of a modified VEGF-A polypeptide or polynucleotide (or a vector comprising such a polynucleotide) to a peripheral tissue in vivo. Preferably, this is achieved by direct injection into the peripheral tissue, or by introduction into-a blood vessel that supplies the peripheral tissue.
Also provided in the present invention are methods of increasing vascular permeability comprising administering a therapeutic amount of a pharmaceutical composition comprising a 6b-modified VEGF-A protein, preferably VEGF-A138, VEGF-A162, or VEGF-A182 in a suitable carrier.
Also provided in the present invention are methods of treating a patient suffering from a wound comprising administering a therapeutic amount of a pharmaceutical composition comprising a 6b-modified VEGF protein preferably VEGF-A138 VEGF-A162, or VEGF-A182 in a suitable carrier.
With the foregoing and other objects, advantages and features of the invention that will become hereinafter apparent, the nature of the invention may be more clearly understood by reference to the following detailed description of the invention, the figures, and the appended claims.